Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q02556 (DNA-binding domain)
 6,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534-851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780-786 of UL9. Although the corresponding region of the homologous protein encoded by varicell-azoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.
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PMID:Identification of residues within the herpes simplex virus type 1 origin-binding protein that contribute to sequence-specific DNA binding. 945 91

The thermodynamics of the c-Myb DNA-binding domain (R2R3) interaction with its target DNA have been analyzed using isothermal titration calorimetry and amino acid mutagenesis. The enthalpy of association between the standard R2R3, the Cys130 mutant substituted with Ile, and the cognate DNA is -12.5 (+/- 0.1) kcal mol-1 at pH 7.5 and at 20 degrees C, and this interaction is enthalpically driven throughout the physiological temperature range. In order to understand the DNA recognition mechanism, several pairs of interactions were investigated using single and multiple-base alterations with single and multiple-amino acid substituted mutants. The interactions between the standard R2R3 and many non-cognate DNAs were accompanied by binding enthalpy changes and heat capacity changes, although their affinities were reduced. The roles of the electrostatic interactions in binding to the cognate and the non-cognate DNAs were also analyzed from the dependency of the thermodynamic parameters on the salt concentration. The heat capacity change was found to be significantly dependent upon the salt concentration. Several mutant proteins bound to the multiple-base altered DNA with very small enthalpy changes, although they bound to the cognate and the single-base altered DNAs with detectable enthalpy and heat capacity changes. From the thermodynamic cycles derived from the DNA binding of the amino acid substituted R2R3 to the base substituted DNA duplexes, the individual thermodynamic mechanisms of the specific DNA recognition of R2R3 were dissected. The local folding mechanism was highlighted by the substitution of Pro with either Gly or Ala at the linker between R2 and R3. The characteristic thermodynamic features of specific and non-specific DNA binding are discussed.
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PMID:Thermodynamics of specific and non-specific DNA binding by the c-Myb DNA-binding domain. 955 Oct 98

Application of the "essential dynamics" method to the NMR cluster of structures for the R2R3 DNA-binding domain of the mouse c-Myb transcriptional activator is described. Using this method, large concerted fluctuations of atoms are extracted showing a hinge-bending motion between the two (R2 and R3) Myb repeats on the basis of NMR data alone. Molecular dynamics simulation of the same protein allowed quantitative comparison of the large concerted motions calculated from experimental and theoretical data, showing a significant degree of similarity. Detailed inspection of the motions reveals a conserved proline that plays a key role in determining hinge flexibility. The proline-to-alanine mutation at this position, which has previously been characterized biochemically, was subjected to molecular dynamics and subsequent essential dynamics analysis. The hinge-bending motion between the two repeats was found to be enhanced for the mutant. The approach described should have general applications, predicting the effect of mutations on protein dynamic properties of other proteins.
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PMID:Essential dynamics from NMR clusters: dynamic properties of the Myb DNA-binding domain and a hinge-bending enhancing variant. 957 Oct 87

We have previously demonstrated a substitutional mutation (glycine to alanine at position 820) of the androgen receptor (AR) gene in a patient with complete androgen insensitivity syndrome (CAIS). We first examined whether the mutation could lead to a disorder in AR binding activity in in vitro expression experiments. In a luciferase assay, the effect of the mutant AR on a target's gene was definitely impaired. However, the mutant AR had less thermal instability compared to that of the patient's fibroblast cell lines established in a whole-cell binding assay. In order to analyze the cause of the thermal instability, a further analysis of exon A in the AR gene was performed because the previous study had been performed only between exon B and H encoding the DNA-binding domain and the hormone-binding domain. The second mutation (leucine to proline at position 257) was newly identified. In in vitro expression experiments, the AR with both mutations showed marked thermal instability, whereas the AR with a mutation in exon A had no effect on thermal stability. The results show that the N-terminal domain might also play an important role in amplifying or modifying the AR binding activity.
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PMID:One additional mutation at exon A amplifies thermolability of androgen receptor in a case with complete androgen insensitivity syndrome. 961 Apr 19

Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfbeta, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.
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PMID:Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta. 963 4

Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation. Many of these zinc-finger domains are involved in DNA binding. This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition. (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition. The protein-DNA interaction was examined in greater detail by using a methylation-interference assay. The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide. This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers.
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PMID:Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition. 963 66

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys-->Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.
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PMID:In vitro analysis of the zinc-finger motif in human replication protein A. 988 30

Studies of yeast DNA topoisomerase II with various alanine-substitution mutations provide strong biochemical support of a recent hypothesis that the type IA and IIA DNA topoisomerases act similarly in their cleavage and rejoining of DNA. DNA breakage and rejoining by either a type IA or a type IIA enzyme are shown to involve cooperation between a DNA-binding domain containing the active-site tyrosine and a Rossmann fold containing several highly conserved acidic residues. For a homodimeric type IIA enzyme, cooperation occurs in trans: the active-site tyrosine in the DNA-binding domain of one protomer cooperates with several residues in the Rossmann fold as well as other regions of the other protomer.
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PMID:Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases. 992 62

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR-->AAA and R290QQ-->AAA), two in the DNA-binding domain (E437E-->AA and V466VV-->AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/triangle up53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, triangle up53C. Stable expression of either the WKR or triangle up53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3-dependent proliferation and granulocyte colony-stimulating factor (G-CSF)-dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF-dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.
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PMID:Dominant negative mutants implicate STAT5 in myeloid cell proliferation and neutrophil differentiation. 1036 Nov 13

Despite the wide spectrum of androgen receptor (AR) mutants described in androgen insensitivity syndromes (AIS), their influence on transactivating and, in particular, transrepressing functions of AR are poorly defined. Rat AR mutants with substitutions in the DNA-binding domain, corresponding to several mutations in AIS patients, were examined for these activities. AR variants (G551V and C562G) with mutations in the first zinc finger (ZF) exhibited reduced DNA binding activity and attenuated transactivation. An R590Q substitution in the second ZF diminished transcriptional activity only from a promoter with a single androgen response element, whereas activation at multiple androgen response element sites was unaffected, despite the poor DNA-binding affinity of R590Q. Another substitution in the second ZF, A579T, yielded similar findings. In comparison to wild-type AR, G551V, and C562G variants had markedly reduced ability to repress an NF-kappaB/RelA-activated promoter but R590Q behaved like the native receptor. AP1 function was repressed not only by wild-type AR but also by the transactivating mutants A579T and R590Q as well as by the transcriptionally inactive mutants G551V and C562G. Furthermore, a Lys-to-Ala substitution in codon 563 of the first ZF switched AR into a ligand-dependent activator at AP1 sites but maintained the ability to repress NF-kappaB/RelA function. Taken together, DNA-binding domain mutations in AIS patients influence transcriptional activating and repressing functions of AR in a selective fashion, which probably contributes to the complexity in the presentation of the AIS phenotype.
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PMID:Transcription activating and repressing functions of the androgen receptor are differentially influenced by mutations in the deoxyribonucleic acid-binding domain. 1038 2


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